The invention relates to novel hemoglobin compositions, particularly novel recombinant mutant hemoglobin compositions, which eliminate or substantially reduce 1) the creation of heart lesions, 2) gastrointestinal discomfort, 3) pressor effects, and 4) endotoxin hypersensitivity associated with the administration of extracellular hemoglobin compositions in various therapeutic applications. Applications described include use as a blood substitute, volume expander and/or oxygen carrier in various therapeutic treatments.
Hemoglobin (Hb) is the oxygen-carrying protein of blood, comprised of four associated polypeptide chains that bear prosthetic groups known as hemes. About 92% of adult human hemoglobin is composed of two alpha globin subunits (xcex11, xcex12) and two beta, globin subunits (xcex21, xcex22) that associate noncovalently to form xcex12 xcex22, commonly known as hemoglobin A0 (WO 93/09143). However, adult hemoglobin may also comprise delta globin subunits. The delta globin subunit replaces beta globin and pairs with alpha globin as alpha2delta2 to form hemoglobin A2. In addition to the globin subunits of adult hemoglobin, a number of hemoglobin subunits are expressed in nature only during embryonic and fetal development including, gamma globin, zeta globin, and epsilon globin. To form embryonic or fetal hemoglobin, zeta globin may replace alpha globin and epsilon and gamma globin may replace beta globin (e.g. to form tetrameric hemoglobin such as alpha2epsilon2, alpha2gamma2, zeta2epsilon2, and zeta2gamma2). Embryonic hemoglobin confers a biological advantage to the developing fetus because it generally has a higher oxygen affinity relative to adult hemoglobin and thus, facilitates fetal oxygen uptake from the maternal blood stream. The structure of hemoglobin is well known and described in Bunn and Forget, eds., Hemoglobin: Molecular, Genetic and Clinical Aspects (W. B. Saunders Co., Philadelphia, Pa.: 1986) and Fermi and Perutz xe2x80x9cHemoglobin and Myoglobin,xe2x80x9d in Phillips and Richards, Atlas of Molecular Structures in Biology (Clarendon Press: 1981).
Expression of various recombinant hemoglobins containing naturally-occurring and non-naturally occurring globin mutants has been achieved. Such methods include the expression of individual globins in recombinant cells, as described, for example, in U.S. Pat. No. 5,028,588, and co-expression of alpha and beta globins in the same cell, as described in U.S. Pat. No. 5,545,727. In addition, di-alpha globin expression, wherein two alpha globins are joined with a short polypeptide linker through genetic fusion and are later coupled with two beta globins to produce a pseudotetrameric hemoglobin molecule, has been described in U.S. Pat. No. 5,545,727 and Looker et al., Nature 356:258-260 (1992). Other modified recombinant hemoglobins are disclosed, e.g., in U.S. Pat. No. 5,844,090.
Solutions of extracellular hemoglobin have been demonstrated to have many therapeutic uses. U.S. Pat. Nos. 5,658,879 and 5,679,638 describe the administration of stroma-free purified wildtype hemoglobin to cancer patients in order to enhance the effects of chemotherapy or radiation therapy. U.S. Pat. No. 5,614,490 describes the use of stroma-free diaspirin crosslinked hemoglobin to increase the perfusion of tissues to treat stroke and ischemia, and to treat hypovolemic, cardiogenic, and septic shock. U.S. Pat. No. 5,428,007 describes the use of recombinant mutant hemoglobin with altered oxygen affinity to increase tissue oxygenation in order to treat burn victims. U.S. Pat. No. 5,631,219 teaches the use of recombinant mutant hemoglobin with altered oxygen affinity to treat anemias, cytopenias, and cachexia, and to stimulate hematopoiesis. And, WO 98/17289 describes the use of stroma-free diaspirin crosslinked hemoglobin to treat head injuries in mammals. The use of crosslinked oxyhemoglobin to treat sickle cell disease is described by Walder et al., J. Mol. Bio., vol. 141, 195-216 (1980).
Nitric oxide acts as a chemical messenger in the control of many important processes in vivo, including neurotransmission, inflammation, platelet aggregation, and regulation of gastrointestinal and vascular smooth muscle tone. The biological actions of nitric oxide are mediated by binding to and activation of soluble guanylyl cyclase, which initiates a biochemical cascade resulting in a variety of tissue-specific responses (Feldman et al., Chem. Eng. News Dec. 26-38 (1993)).
Elucidating the functions of nitric oxide has depended largely on inhibition of the nitric oxide-generating enzyme, nitric oxide synthase. Most conclusions about the effects of cell-free hemoglobin have been drawn based on experiments involving nitric oxide synthase inhibitors and/or nitric oxide donors. While the rapid, high-affinity binding of nitric oxide to deoxyhemoglobin is well known, the importance of the oxidative reaction between nitric oxide and oxyhemoglobin is not as widely appreciated. In this reaction, the nitric oxide molecule does not bind to the heme, but reacts directly with the bound oxygen of the oxyhemoglobin complex to form methemoglobin and nitrate (Doyle et al., J. Inorg. Biochem. 14: 351-358 (1981)). The chemistry is analogous to the rapid reaction of nitric oxide with free superoxide in solution (Huie et al., Free Rad. Res. Comms. 18: 195-199 (1993)). Both the heme iron and nitric oxide become oxidized by the bound oxygen atoms, and the reaction occurs so rapidly that no replacement of oxygen by nitric oxide is observed (Eich et al., infra.).
Since nitric oxide is produced and consumed on a continuous basis, there is a natural turnover of nitric oxide in vivo. When a cell-free hemoglobin is administered, the balance between nitric oxide production and consumption is altered by reactions with hemoglobin. The most relevant parameter for nitric oxide scavenging by hemoglobin is the rate of reaction with nitric oxide, not the position of the hemoglobin allosteric (R/T) equilibrium. The oxidative reaction is irreversible, and nitric oxide binding to deoxyhemoglobin is effectively irreversible on physiologic timescales since the half-life for dissociation of nitrosylhemoglobin is 5-6 hours (Moore et al., J. Biol. Chem. 251: 2788-2794 (1976).
When nitric oxide molecules react with oxyhemoglobin or deoxyhemoglobin, they are eliminated from the pool of signal molecules. Once sufficient nitric oxide molecules are eliminated, it is believed, certain adverse conditions are created. For example, hemoglobin can bind nitric oxide causing the prevention of vascular relaxation and potentially leading to hypertension that is sometimes observed after administration of certain extracellular hemoglobin solutions. In addition, the ability of nitric oxide to oxidize oxyhemoglobin producing nitrate and methemoglobin could also lower free concentrations of nitric oxide and lead to hypertension.
Nitric oxide is also needed to mediate certain inflammatory responses. For example, nitric oxide produced by the endothelium inhibits platelet aggregation. Consequently, as nitric oxide is bound by cell-free hemoglobin, platelet aggregation may be increased. As platelets aggregate, they release potent vasoconstrictor compounds such as thromboxane A2 and serotinin. These compounds may act synergistically with the reduced nitric oxide levels caused by hemoglobin scavenging resulting in significant vasoconstriction. In addition to inhibiting platelet aggregation, nitric oxide also inhibits neutrophil attachment to cell walls, which in turn may lead to cell wall damage.
Several undesirable side effects have been observed by applicants upon administering solutions of extracellular wild-type human hemoglobin to test subjects. Applicants"" experimental results in sensitive test animals, as described in detail below, demonstrate that extracellular hemoglobin compositions such as those including wild-type adult human hemoglobin molecules administered at therapeutic dosages result in the formation of myocardial necrosis in heart tissue. While not being bound to a particular theory, applicants believe that there is a direct correlation between nitric oxide (NO) scavenging by these extracellular hemoglobin molecules and the incidence of myocardial necrosis in treated animals. Applicants found that administration of nitric oxide synthase (NOS) inhibiting drugs to test animals generates heart lesions similar to those observed with the administration of extracellular hemoglobin compositions.
In addition, several other undesirable side effects have been observed by others with the use of previously described hemoglobins. Mild hypertension has sometimes been observed following administration of certain extracellular hemoglobin solutions. It is believed by many that the hypertension is due to depletion of nitric oxide in the wall of the vasculature, based in part on the known high affinity of deoxyhemoglobin for nitric oxide (Schultz et al., J. Lab. Clin. Med.122:301-308 (1993); Thompson et al., J. Appl. Physiol. 77:2348-2354 (1994); Rooney et al., Anesthesiology 79:60-72 (1993)). Extravasation of the hemoglobin into endothelial cells or interstitial spaces may cause significant consumption of nitric oxide (Gould et al., World J. Surg. 20: 1200-1207 (1996)). A recent study also suggests that the oxidative reaction of nitric oxide with the bound oxygen of oxyhemoglobin may be of greater significance in vivo than simple binding to the iron atom as reported in Eich et al., Biochemistry 35: 6976-6983 (1996). Eich et al. showed that steric hindrance introduced by substitution of amino acids adjacent to bound oxygen can markedly lower the rate of nitric oxide-induced oxidation.
Transient mild to moderate gastrointestinal effects are another side effect that has been commonly observed with the administration of extracellular hemoglobin compositions (Tsuchida, et al., introduction, Artificial Red Cells: Materials, Performances and Clinical Study as Blood Substitutes. Liely, New Your, N.Y., p. 1-20 (1995), and Viele, et al., Anesthesiology, 86: 848-58 (1997)). These effects include upper gastrointestinal discomfort including mid-epigastric discomfort, abdominal pain and/or dyspepsia and/or lower gastrointestinal discomfort including lower abdominal pain, flatulence and/or diarrhea. The gastrointestinal events typically develop one to three hours post infusion, last from one to several hours, and wax and wane over time. Nitric oxide is known to be an important modulator of the proper function of the smooth muscle tissue which regulates gastrointestinal contractility. Observed gastrointestinal effects are thus thought to be due in part to gastrointestinal dysmotility caused by hemoglobin reactivity with nitric oxide (Conklin, et al., J. Pharmacol. Exp. 273: 762-67 (1995), and Conover, et al., Life Sci. 59: 1861-69 (1995).).
In addition, endotoxin hypersensitivity has also been observed in animals which are co-administered extracellular hemoglobin solutions and low doses of lipopolysaccharide. These animals usually die from septic shock within 48 hours of co-administration.
Thus, the need exists for a method of eliminating or substantially reducing the creation of heart lesions in mammals treated with the extracellular hemoglobin used in these applications, as well as other deleterious side effects, such as pressor effects or gastric discomfort, associated with such use. Accordingly, a need exists for hemoglobin compositions which eliminate or substantially reduce the occurrence of heart lesions and other adverse conditions, while still functioning as an effective oxygen carrying agent.
One problem which has been encountered in storing purified extracellular hemoglobin solutions for therapeutic use is the formation of hemoglobin aggregates and precipitates over time. Addition of antioxidants, such as n-acetylcysteine, dihydrolipoic acid, or ascorbate, is known to prevent precipitate formation. However, chemical-free methods of preventing hemoglobin aggregate formation are preferred to simplify regulatory approval of the hemoglobin solutions.
The invention relates to hemoglobins which reduce (reduced lesion) or eliminate heart lesions (lesion free) when administered as a therapeutic agent. These hemoglobins are preferably recombinantly produced mutant hemoglobins, which may be chemically crosslinked. Therefore, in one aspect, the invention is directed to pharmaceutical compositions comprising recombinant hemoglobins which cause reduced or no heart lesions in a suitable pharmaceutical carrier.
The invention also relates to hemoglobins with moderately reduced (reduced gastrointestinal effect) or greatly reduced deleterious gastrointestinal effects (low gastrointestinal effect) when administered as a therapeutic agent. These hemoglobins are preferably recombinantly produced mutant hemoglobins, which may be chemically crosslinked. Therefore, in another aspect, the invention is directed to pharmaceutical compositions comprising recombinant hemoglobins which cause moderately or greatly reduced gastrointestinal effects in a suitable pharmaceutical carrier.
The invention also relates to hemoglobins with moderately reduced (reduced pressor effect) or greatly reduced pressor effects (low pressor effect) when administered as a therapeutic agent. These hemoglobins are preferably recombinantly produced mutant hemoglobins, which may be chemically crosslinked. Therefore, in another aspect, the invention is directed to pharmaceutical compositions comprising recombinant hemoglobins which cause moderately or greatly reduced pressor effects in a suitable pharmaceutical carrier.
The invention also relates to hemoglobins which cause reduced endotoxin hypersensitivity effects (reduced endotoxin effect) when administered as a therapeutic agent. These hemoglobins are preferably recombinantly produced mutant hemoglobins, which may be chemically crosslinked. Therefore, in another aspect, the invention is directed to pharmaceutical compositions comprising recombinant hemoglobins which cause reduced endotoxin hypersensitivity effects in a suitable pharmaceutical carrier.
The invention also relates to hemoglobins which have any combination or permutation of the above biological characteristics, in either a reduced or low to zero degree. Therefore, in another aspect, the invention is directed to pharmaceutical compositions comprising recombinant hemoglobins which cause a combination of the above effects in a suitable pharmaceutical carrier.
The present invention is also directed to the use of any of the above hemoglobins in a physiologically acceptable solution as an oxygen carrying whole blood substitute.
The present invention is also directed to the use of any of the above hemoglobins to treat hypovolemia, and to resuscitate a mammal suffering from hemorrhagic or cardiogenic shock.
Another aspect of the present invention is the use of any of the above hemoglobins to increase tissue perfusion in order to treat ischemia or stroke, and to increase oxygenation of tissues which have been subject to physical damage, such as wounds and burns.
Another aspect of the present invention is the use of any of the above hemoglobins to treat anemia and to stimulate hematopoiesis.
Another aspect of the present invention is the use of any of the above hemoglobins to treat head injury in a mammal.
Another aspect of the invention is directed to the administration of a cancer therapy enhancing amount of any of the above hemoglobins to a patient undergoing radiation or chemotherapy in order to enhance the tumor-reducing effects of that therapy.
Yet another aspect of the present invention is directed to the use of any of the above hemoglobins to treat sickle cell anemia patients, in particular such patients undergoing vaso-occlusive crisis.
In addition to hemoglobin compositions with novel biological properties, as described above, the present invention is also directed to novel recombinant mutant hemoglobins which demonstrate a reduced rate of reactivity with nitric oxide. Specifically, the invention is directed to recombinant mutant hemoglobins comprising a mutation or mutations in the a subunit corresponding to B10(Leuxe2x86x92Tyr); B10(Leuxe2x86x92Trp)+E11(Valxe2x86x92Phe)+G8(Leuxe2x86x92Ala); B10(Leuxe2x86x92Trp)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Met)+G8(Leuxe2x86x92Val); B10(Leuxe2x86x92Trp)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Phe)+G8(Leuxe2x86x92Val); B10(Leuxe2x86x92Trp)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Leu)+G8(Leuxe2x86x92Val); B10(Leuxe2x86x92Phe)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Met)+G8(Leuxe2x86x92Phe); B10(Leuxe2x86x92Phe)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Met)+G8(Leuxe2x86x92Met); B10(Leuxe2x86x92Phe)+E7(Hisxe2x86x92Gln)+E11(Valxe2x86x92Met)+G8(Leuxe2x86x92Ile); or B9(Alaxe2x86x92Phe). The invention is also directed to recombinant mutant hemoglobins comprising a mutation or mutations in the xcex2 subunit corresponding to B9(Alaxe2x86x92Leu)+E11(Valxe2x86x92Met)+G8(Leuxe2x86x92Trp); B9(Alaxe2x86x92Leu)+E11(Valxe2x86x92Leu)+G8(Leuxe2x86x92Trp); B9(Alaxe2x86x92Leu)+E11(Valxe2x86x92Leu); B9(Alaxe2x86x92Phe)+E11(Valxe2x86x92Leu); B9(Alaxe2x86x92Trp)+E11(Valxe2x86x92Leu); B9(Alaxe2x86x92Phe); B9(Alaxe2x86x92Trp); B9(Alaxe2x86x92Leu); B9(Alaxe2x86x92Met); E11(Valxe2x86x92Met); B10(Leuxe2x86x92Phe)+B14(Leuxe2x86x92Phe); B9(Alaxe2x86x92Leu)+G8(Leuxe2x86x92Trp); B9(Alaxe2x86x92Trp)+G8(Leuxe2x86x92Phe); B9(Alaxe2x86x92Phe)+G8(Leuxe2x86x92Trp); B9(Alaxe2x86x92Trp)+G8(Leuxe2x86x92Trp); B10(Leuxe2x86x92Ala)+E7(Hisxe2x86x92Phe)+G8(Leuxe2x86x92Trp); or E11(Valxe2x86x92Trp)+G8(Leuxe2x86x92Trp). In addition, the invention is also directed to recombinant hemoglobin mutants comprising a combination of any of the preceding mutations in both xcex1 and xcex2 subunits.
In addition, the present invention is also directed towards recombinant mutant hemoglobins which exhibit decreased aggregate formation when stored in solution in deoxygenated form for prolonged periods of time, and which do not comprise the sickle cell mutation, xcex26 Gluxe2x86x92Val. Preferably, such hemoglobins comprise a mutation to a hydrophilic or bulky amino acid in the xcex2 subunit at the 87 threonine, and comprise the native amino acid xcex26 Glu. More preferably, such hemoglobins comprise a mutation chosen from the group consisting of xcex287 Thrxe2x86x92Asp, Glu, Arg, Lys, His, Tyr, Gln, or Trp. Even more preferably, such hemoglobins comprise a mutation chosen from the group consisting of xcex287 Thrxe2x86x92Asp, Gln, Glu, Arg, Lys, or His. Most preferably, such hemoglobins comprise the mutation xcex287 Thrxe2x86x92Gln.